Structure of the mitochondrial import gate reveals distinct preprotein paths

Abstract

The translocase of the outer mitochondrial membrane (TOM) is the main entry gate for proteins1,2,3,4. Here we use cryo-electron microscopy to report the structure of the yeast TOM core complex5,6,7,8,9 at 3.8-Å resolution. The structure reveals the high-resolution architecture of the translocator consisting of two Tom40 β-barrel channels and α-helical transmembrane subunits, providing insight into critical features that are conserved in all eukaryotes1,2,3. Each Tom40 β-barrel is surrounded by small TOM subunits, and tethered by two Tom22 subunits and one phospholipid. The N-terminal extension of Tom40 forms a helix inside the channel; mutational analysis reveals its dual role in early and late steps in the biogenesis of intermembrane-space proteins in cooperation with Tom5. Each Tom40 channel possesses two precursor exit sites. Tom22, Tom40 and Tom7 guide presequence-containing preproteins to the exit in the middle of the dimer, whereas Tom5 and the Tom40 N extension guide preproteins lacking a presequence to the exit at the periphery of the dimer.

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Fig. 1: Overall structure of the TOM complex and bound phospholipid.
Fig. 2: Tom5, Tom6 and the C-terminal segment of Tom40.
Fig. 3: N extension of Tom40.
Fig. 4: The N extension of Tom40 recruits Mia40 and promotes import of MIA substrates.
Fig. 5: The N extension of Tom40 plays a dual role in the MIA import pathway.

Data availability

The cryo-EM density maps have been deposited in the Electron Microscopy Data Bank under accession code EMD-9851. The atomic coordinates have been deposited in the Protein Data Bank under accession code 6JNF. All the data from this study are available in the Article, its Extended Data and Supplementary Information. The uncropped blots, gels and plates are provided in Supplementary Fig. 2.

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Acknowledgements

We thank the members of the Endo and Pfanner labs for discussion and critical comments on the manuscript. This work was supported by JSPS KAKENHI to T.E. (15H05705 and 2222703), K.I. (16K21680 and 18K11543), T.S. (19K16077 and 18KK0197) and T.A. (26119003) and JST CREST grants to T.E. (JPMJCR12M1) and to M.K. (JPMJCR14M1); a European Research Council (ERC) Consolidator grant to N.W. (no. 648235); Deutsche Forschungsgemeinschaft grants to T.B. (BE 4679/2-2) and N.P. (PF 202/9-1); and Germany’s Excellence Strategy to T.B., N.W. and N.P. (CIBSS – EXC-2189—Project ID 390939984; GSC-4 Spemann Graduate School). The following grants are also acknowledged: a grant from Takeda Science Foundation (to T.E.); Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under grant numbers JP19am01011115 (to M.K.) and JP19am0101114 (to K.I.); grants from the Ichiro Kanehara Foundation for the Promotion of Medical Sciences and Medical Care, Waksman Foundation of Japan, Tokyo Biochemical Research Foundation, Sumitomo Foundation, Naito Foundation, and Uehara Memorial Foundation (to T.S.); and the Australian Research Council Discovery Project DP160100227 (to T.L.). We acknowledge the Cryo-EM Facility of the University of Tokyo. Y.A. was supported by a Research Fellowship for Young Scientists from the Japan Society of the Promotion of Science (15J07687). Work included in this study has also been performed in partial fulfillment of the requirements for the doctoral thesis of C.L. at the University of Freiburg. We thank T. Nishizawa for discussion and advice on cryo-EM data processing, M. H. Schuler for analysis of yeast mutants, R. Beckmann and A. Heuer for advice on cryo-EM measurements and H. Nishitoh and Y. Inaba for instruments and reagents for T.S.

Author information

Y.A. performed most of the experiments and wrote the paper. A.T. performed and M.K. supervised cryo-EM measurements and data processing. J.Q., J. Song, C.L., L.-S.W., M.W. and C.S. performed functional studies on the role of TOM proteins in preprotein import into mitochondria, characterized yeast mutants, studied the import of precursor proteins into isolated mitochondria and analysed data. K.I. performed bioinformatics analyses and wrote a part of the paper. T.S. performed crosslinking and T.L. designed and supervised crosslinking and wrote a part of the paper. H.S. analysed yeast cell growth of double mutants of tom5Δ and Tom40 N-terminal truncation. K.Y., S.K. and J. Suzuki assisted with designing preparation of the materials including the yeast TOM complex. H.A. and T.A. performed HS-AFM measurements. N.W., T.B. and N.P. designed and supervised functional studies on the role of TOM proteins in preprotein import into mitochondria, analysed data and wrote a part of the paper. T.E. designed the research and wrote the paper. All authors discussed the results of the experiments and commented on the manuscript.

Correspondence to Toshiya Endo.

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Extended data figures and tables

Extended Data Fig. 1 Purification of the TOM complex.

a, Forms of the TOM complex. Newly imported Tom40 takes a β-barrel structure and forms an assembly ‘dimer’ lacking Tom22 and Tom20. The dimer is matured into the ‘trimer’ with the receptors Tom22 and Tom204. Even after the maturation, the trimer dynamically exchanges with a minor population of the assembly dimer4. After solubilization of mitochondria, the TOM complex can be found as a trimer, containing Tom20 at least partly. Our previous in vivo crosslinking analyses revealed the subunit arrangement of the trimer4,32, and the present study revealed the high-resolution cryo-EM structure of the dimeric TOM core complex that contains Tom22, but lacks Tom20. b, Elution profile of gel filtration of the purified TOM complex. c, Blue native electrophoresis gel of the TOM complex stained with Coomassie brilliant blue. The apparent molecular weight of the TOM complex is ~440 kDa on blue native electrophoresis, which corresponds to the trimer. d, SDS–PAGE gel of the TOM complex stained with Coomassie brilliant blue. The bands for Tom40, Tom22–His10, Tom20 and Tom5 were assigned by western blotting with antibodies against each subunits. The presence of Tom5, Tom6 and Tom7 were confirmed by mass spectrometry (MS) analyses. The bands for Tom5 and Tom7 were assigned by MS/MS analyses of the tryptic fragments of the proteins contained in each band. e, MALDI–TOF MS of purified TOM complex in the m/z range below 10,000. Calculated molecular weights of yeast small TOM subunits are 5986.0 (Tom5), 6407.6 (Tom6), and 6871.0 (Tom7). Tom7 appears to lack the N-terminal Met, and Tom6 may be acetylated. The data are representative of six (b, c), four (d) and two (e) independent experiments. f, Typically observed particles of HS-AFM images of the TOM complex trimer and dimer on mica.

Extended Data Fig. 2 Cryo-EM analysis of the TOM complex.

a, A micrograph of the TOM complex. b, Selected 2D classes showing various orientations. c, FSC curve after the application of soft mask in RELION, showing FSC of 0.143. d, The schematic workflow of the steps followed in data processing leading to the final structure of the TOM core complex. e, Angular particle distribution in the final reconstruction. f, Local map resolution of the final TOM complex structure with colours according to the scale on the right side. g, FSC curve between the EM map and the atomic model of the TOM complex. h, An example of the fit of the model into the density map; the region around β-strands 11–18 of Tom40 is shown.

Extended Data Fig. 3 β-barrel structure of Tom40.

a, Hydrogen bonds between the 19 β-strands on the basis of the EM structure. Circles, pentagons and squares indicate residues in loops, α-helices and β-strands, respectively. Disordered regions are coloured in grey. Hydrogen bond, CO–NH distance < 4.0 Å; weak hydrogen bond, 4.0 Å ≤ CO–NH distance ≤ 4.5 Å; no hydrogen bond, 4.5 Å < CO–NH distance. b, Relative positions of the other TOM subunits and phospholipid in contact with the Tom40 β-barrel. c, Summary of previous crosslinking results of Tom404. BPA-incorporated residues are labelled in bold. Crosslinked partners are indicated by different colours. Residues labelled with a red asterisk are not within a distance of 5 Å from their crosslinked partners in the EM structure.

Extended Data Fig. 4 Model for transition between the Tom22-containing dimer and trimer.

a, Comparison of the cryo-EM structure of Tom22-containing dimer (D) and crosslinked-based structure model of Tom22-containing trimer (T). b, The residues whose proximity relationships are inconsistent between the trimer (crosslinking) and the dimer (EM structure) are mapped on the EM structure. Top view from the cytosolic side (left) and side view (right) of the EM dimer structure (cartoon model) with the side chains (space-filling model) of the residues that were crosslinked to other subunits4 but were not within a distance of 4.5 Å from the crosslinked partners in the EM structure (Supplementary Table 1), and therefore judged to be important for formation of the mature trimer4. c, The residues suggesting the possible difference in the subunit interfaces between the trimer and dimer are mapped on the EM structure. Although Tom22A P112 and Tom40B Y309 face each other in the EM structure, the Tom22 transmembrane helix was crosslinked to two Tom40 molecules through Tom22 P112 and Tom40 Y3094, suggesting that these two residues should not face each other in the trimer. Tom22 E120 and Tom40 R310 are evolutionary conserved (Extended Data Fig. 7 and Supplementary Table 2) and electrostatic interaction between the two residues would contribute to stabilization of the dimer. d, e, Upon formation of the trimer from the dimer, the Tom22 transmembrane helix in the EM structure may rotate (with a possible conformational change), so that Tom22A P112 faces the Tom40A molecule and Tom22A S114 interacts with the Tom40B molecule. This rotation would lead to a switch of the electrostatic-interaction pairs from Tom22 E120 and Tom40 R310 to Tom22 E120 and Tom40 K90 with possible concomitant movement of the Tom7 C-terminal segment; the second short C-terminal helix of Tom7 appears to be stabilized by the hydrogen bond between the carbonyl O of L51 in Tom7 and the side-chain NH of K90 in Tom40 in the dimer, which is probably lost in the trimer. The rotation of the Tom22 transmembrane helix would also allow K94 and R89 of Tom22 to interact with D350 of the neighbouring Tom40 molecule. Basic and acidic residues are coloured in blue and red, respectively.

Extended Data Fig. 5 Conserved interactions between the Tom40 β-barrel and the α-helical subunits.

ad, Interactions are shown between the Tom40 β-barrel and the α-helical subunits Tom22 (a), Tom7 (b), Tom6 (c) and Tom5 (d). Tom40 residues of the β-barrel outer wall and loops are shown in space-filling form and coloured by evolutionary conservation (darker blue represents more conserved residues). Tom40 residues at ‘conserved amino acid’ (conserved) positions within 4.5 Å distance of the α-helical subunits are labelled. The main chains of the α-helical subunits are shown in ribbon form, and residues at conserved positions within 4.5 Å distance of Tom40 are shown in stick form, labelled, and coloured by evolutionary conservation (deeper colour represents more highly conserved residues). Residues in parentheses are the most frequently observed residue at each conserved position. Pairs of residues at conserved positions of Tom40 β-barrel and α-helical subunits in proximity (≤4.5 Å) are underlined and bolded: Tom40B R310 (R) and Tom22 E120 (E), Tom40B S312 (S) and Tom22 S116 (A), Tom40B Y314 (F) and Tom22 L109 (L), Tom40B V324 (V) and Tom22 L109 (L), Tom40B V324 (V) and Tom22 L108 (L), Tom40B V324 (V) and Tom22 T105 (S), Tom40B L328 (L) and Tom22 P112 (P), Tom40B R330 (K) and Tom22 A119 (E), Tom40A L336 (F) and Tom22 L115 (L), Tom40B H346 (H) and Tom22 T105 (S), Tom40B H346 (H) and Tom22 T104 (T), and Tom40B H346 (H) and Tom22 W100 (W) (a); Tom40 F98 (F) and Tom7 G40 (G) and Tom40 G129 (G) and Tom7 I33 (I) (b); Tom40 L259 (Y) and Tom6 V46 (V), Tom40 L259 (Y) and Tom6 Q50 (Q), Tom40 G299 (G) and Tom6 N38 (S) and Tom40 S320 (S) and Tom6 N38(S) (c). No conserved proximate residue pair was found between Tom40 and Tom5 (d). Phospholipid (modelled as phosphatidylethanolamine) is shown in space-filling form; carbon, nitrogen, oxygen and phosphorus are coloured light green, blue, red and magenta, respectively (a).

Extended Data Fig. 6 Formation of the two exit sites of the TOM channel for different classes of precursor proteins.

a, Disordered regions of Tom22 and Tom40 facing the IMS. b, The TOM complex viewed from the IMS side shows that the trans presequence binding site (orange) is formed by the hydrophilic loop of Tom7 (residues 43–49), the hydrophilic β1–β2 loop (residues 92–96, M94 is disordered) and C-terminal domain (residues 363–387, residues after L374 are disordered, G363–E366 form a loop and E367–G373 form a helix) of Tom40 and the acidic IMS domain of Tom22 (residues 120–152, residues after 131 are disordered, the transmembrane helix is extended up to F130). Disordered parts are shown with beads. c, Crosslinked products of Tom22 containing BPA at the indicated positions were detected by the indicated antibodies. Crosslinked products between Tom22 and Tim50 (22–50) are indicated. Representative data of two independent experiments. d, Radiolabelled Oxa1 precursor was imported into wild-type and Tom40(Δ364–387) mitochondria for the indicated periods in the absence or presence of a membrane potential (Δψ) and analysed by blue native electrophoresis and autoradiography. Representative data of three independent experiments. Quantification of Oxa1 bound to the TOM complex; mean values ± s.e.m. (n = 3 biologically independent experiments); the amount of bound Oxa1 in wild-type mitochondria after 20 min (−Δψ) was set as 100% (control). e, Mitochondria isolated from wild-type and Tom40(Δ364–387) yeast grown at 30 °C were analysed by SDS–PAGE and western blotting using the indicated antisera. Representative data of four independent experiments. Amount of total mitochondrial protein is indicated. f, Mitochondria isolated from wild-type and Tom40(Δ364–387) yeast grown at 30 °C were analysed by blue native electrophoresis and western blotting with Tom40 antiserum. Representative data of four independent experiments. g, Radiolabelled Tim9 was imported and assembled into the TIM22 complex in wild-type and Tom40(Δ364–387) mitochondria; data are mean ± s.e.m. (n = 3 biologically independent experiments); the amount in wild-type mitochondria after 40 min import was set as 100% (control). h, The trans presequence binding sites for presequence-containing proteins (b) and the N extension of Tom40 recruiting small TIM chaperones (for presequence-lacking β-barrel and carrier proteins) and Mia40 (for MIA substrates) are spatially separated to form distinct precursor exit sites at the outlet of the TOM channel. The two exit sites are connected to the different translocation paths (yellow broken lines) that include either the aligned acidic patches (red) or the aligned hydrophobic patches (green) inside the Tom40 β-barrel pore (Extended Data Fig. 9). Disordered parts are shown with beads. Source data

Extended Data Fig. 7 Roles of Tom5 and the N extension of Tom40 in MIA import and assembly.

ae, Import of radiolabelled Tim9 precursor into isolated wild-type, tom5∆, tom6∆, tom7∆, tom22∆, tom20∆ and tom70∆ mitochondria, analysed by blue native electrophoresis (top) and SDS–PAGE (bottom). TIM22, TIM22 complex; Tim9–Mia40, mixed disulfide intermediate of Tim9 with Mia40; TIM9–10, Tim9–Tim10 complex. Representative data of three (a, b, top), four (d, top) or two (other panels) independent experiments. f, Mitochondria were isolated from wild-type, Tom40(∆58) and Tom40(∆59) yeast cells, which were grown at 30 °C and, where indicated, additionally shifted to 37 °C for 12 h, and analysed by SDS–PAGE and western blotting using the indicated antisera. Representative data of two independent experiments. g, Radiolabelled Tom5 precursor was imported into mitochondria isolated from wild-type and Tom40(∆59) yeast cells, which were grown at 30 °C and shifted to 37 °C for 12 h, followed by incubation with sodium carbonate. The carbonate-resistant pellets were analysed by SDS–PAGE and autoradiography. Representative data of three independent experiments. h, Mitochondria of wild-type, Tom40(∆58) and Tom40(∆59) cells grown at 30 °C were analysed by blue native electrophoresis and western blotting using Tom40- and Mia40-specific antisera. Representative data of two independent experiments. i, Imports of radiolabelled Atp2 and b2(167)∆DHFR into wild-type, Tom40(Δ58) and Tom40(Δ59) mitochondria were analysed by SDS–PAGE. Representative data of three independent experiments. ∆ψ, membrane potential; p, precursor form; m, mature form; i, intermediate form. j, Mitochondria of wild-type, Tom40(∆58) and Tom40(∆59) cells grown at 30 °C were subjected to hypo-osmotic swelling and treatment with proteinase K (Prot. K) as indicated. Proteins were analysed by SDS–PAGE and western blotting using the indicated antisera. Representative data of two independent experiments. The IMS protein Tim13 shows that the outer membranes of (non-swollen) Tom40(∆58) and Tom40(∆59) mitochondria were intact like those of wild-type mitochondria. k, Import of radiolabelled Su9–DHFR precursor (top) or AAC (bottom) into wild-type, Tom40(L57A) and Tom40(L57A/T62A) mitochondria was analysed by SDS–PAGE (top) or blue native gel (bottom). Representative data of three (Su9–DHFR) or two (AAC) independent experiments. l, Mitochondria of wild-type, Tom40(L57A) and Tom40(L57A/T62A) strains grown at 30 °C and, where indicated, additionally shifted to 37 °C for 29.5 h, were analysed by SDS–PAGE and western blotting using the indicated antisera. Representative data of two independent experiments.

Extended Data Fig. 8 MIA import and assembly are disturbed in point mutants of the Tom40 N extension.

a, Wild-type, Tom40(L57A) and Tom40(L57A/T62A) mitochondria from cells grown at 30 °C were analysed by blue native electrophoresis and western blotting using Tom40- and Mia40-specific antisera. Representative data of two independent experiments. b, Wild-type, tom5∆, Tom40(L202A) and Tom40(L57A/T62A) mitochondria were treated with DTT or 4-DPS and the redox state of Mia40 was analysed by non-reducing SDS–PAGE and western blotting. Representative data of three independent experiments. Red, reduced form; oxid., oxidized form. c, Growth analysis of yeast strains expressing the indicated Tom40 variants on YPG. Representative data of two independent experiments. d, Radiolabelled Tim9 was imported into isolated wild-type and Tom40(L202A) mitochondria for 5 min, followed by addition of iodoacetamide (IA), and incubated further as indicated. The import was analysed by blue native electrophoresis. Representative data of two independent experiments. TIM22, TIM22 complex; Tim9–Mia40, mixed disulfide intermediate of Tim9 with Mia40; TIM9–10, Tim9–Tim10 complex. e, Import of radiolabelled Tim9(C55S) into wild-type and Tom40(L202A) mitochondria followed by treatment with proteinase K as indicated. The import was analysed by blue native electrophoresis (top) and non-reducing SDS–PAGE (bottom). Representative data of two independent experiments. Tim9–Mia40, mixed disulfide intermediate of Tim9(C55S) with Mia40. f, Wild-type and Tom40(L202A) mitochondria from cells grown at 30 °C were analysed by blue native electrophoresis and western blotting using Tom40 and Mia40 specific antisera. Representative data of two independent experiments. g, Wild-type and Tom40(L202A) mitochondria from cells grown at 30 °C were treated with proteinase K where indicated. Representative data of two independent experiments. The stability of IMS-exposed proteins indicates that the outer membranes of Tom40(L202A) mitochondria were intact like those of wild-type mitochondria. h, Import of radiolabelled Su9–DHFR (top) and AAC (bottom) into wild-type and Tom40(L202A) mitochondria was analysed by SDS–PAGE (top) or blue native electrophoresis (bottom). Representative data of three independent experiments.

Extended Data Figure 9 The pore interior of Tom40 and precursor import paths.

a, b, The views from the cytosol (top) and cut-away views (bottom) for Tom40. Section planes for the cut-away views are indicated with broken lines in the top panels. The side chains of the Tom40 residues crosslinked to the translocation intermediates of a presequence-containing precursor pSu9–DHFR (a) and of a hydrophobic presequence-lacking precursor AAC–DHFR (b) are shown on the EM structure with colour reflecting the amount of crosslinked products detected4. The acidic patches (red) and hydrophobic patches (green) are shown in the cut-away views for the β-barrel side of the pore interior of Tom40. Possible precursor translocation paths are indicated with arrows by broken lines (bottom). c, Section curved plane for the cut-away views (centre, bottom) is indicated with a broken line in the view from the cytosolic side (top). The acidic patches (red), hydrophobic patches (green) and possible precursor translocation paths (broken lines) are shown (bottom).

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics

Supplementary information

Supplementary Figures

This file contains Supplementary Figures 1 (alignment of the N-terminus to β-strand 14 of Tom40 from various organisms) and 2 (the uncropped version of all the blots, gels and plates in this study).

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Supplementary Tables

This file contains Supplementary Tables 1-4.

Video 1

HS-AFM observation of the purified TOM complex on a mica plate (200 nm × 200 nm).

Video 2

HS-AFM observation of the purified TOM complex scattered on a mica plate showing conversion of the trimer to the dimer (50 nm × 50 nm).

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Araiso, Y., Tsutsumi, A., Qiu, J. et al. Structure of the mitochondrial import gate reveals distinct preprotein paths. Nature 575, 395–401 (2019) doi:10.1038/s41586-019-1680-7

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